Stabilized compositions of aqueous reduced coenzyme q solution

ABSTRACT

The present invention provides a reduced coenzyme Q-containing solution capable of being retained more stably against oxidation, by using a reduced coenzyme Q which is hydrophobic and susceptible to oxidation and thus unstable. The aqueous solution containing a reduced coenzyme Q obtained in the present invention is a solution containing a reduced coenzyme Q and an antioxidant such as vitamin C and/or a chelating agent such as ethylenediaminetetraacetic acid.

TECHNICAL FIELD

The present invention relates to a solution containing a reducedcoenzyme Q as a constituent and, more particularly, to an aqueoussolution containing an antioxidant and/or a chelating agent addedthereto for maintaining a reduced coenzyme Q stable against oxidation.

BACKGROUND ART

Coenzymes Q are essential constituents widely distributed in livingbodies, from bacteria to mammals and are known as mitochondrial electrontransfer system constituents in cells of living bodies. Through repeatedoxidation and reduction in mitochondria, coenzymes Q perform theirfunction as transmitter components in the electron transfer system and,further, reduced coenzymes Q are known to have antioxidant activity. Inhumans, coenzyme Q₁₀, whose coenzyme Q side chain comprises 10 repeatingstructures, is the main component and, generally, about 40 to 90%thereof occurs in reduced form in living bodies. The physiologicalactivities of coenzymes Q may be energy production activation throughmitochondrial activation, cardiac function activation, cell membranestabilizing effect, and cell protecting effect through antioxidantactivity.

Coenzymes Q are known to be useful in various application fields. Forexample, oxidized coenzyme Q₁₀ is used as a remedy for congestive heartfailure owing to its effects on the heart. Besides such medical uses,they are orally used as nutrients or nutritional supplements, likevitamins. However, coenzymes Q are highly liposoluble and hardly solublein water and, therefore, only oral preparations and dermal preparationsare known as their practical uses.

In recent years, various reports have been published about theaggravation of diseases due to increases in oxidative stress in blood.Typical examples are arteriosclerosis, complications of diabetes and thelike diseases. These diseases are caused and/or aggravated bydenaturation of lipids and the like due to various oxidative stressesoccurring in blood. For counteracting such effects of oxidativestresses, antioxidant activity promotion by administration of anantioxidant is effective. Vitamin E is a compound representative of theliposoluble antioxidant substances considered to be more effective ininhibiting lipid peroxidation and is in wide use in disease preventionand so on.

Recently, it has been reported that the coexistence of reduced coenzymeQ₁₀ is important for vitamin E to properly perform its antioxidantactivity (Bowry et al., 1993, J. American Chemical Society, 115,6029-6044), and the importance of coenzymes Q as liposoluble antioxidantsubstances is becoming clear.

Coenzymes Q have themselves strong antioxidant activity and, therefore,the antioxidant activity in blood can be effectively enhanced by sendinga sufficient amount of reduced coenzymes Q in solubilized form intoblood. The enhanced antioxidant activity in blood is considered to beuseful widely in preventing vascular lesions duringischemia-reperfusion, preventing restenosis in arteriosclerosis,preventing vascular lesions following cerebral infarction, preventingarteriosclerosis, preventing complications of diabetes, and preventing anumber of other diseases from being aggravated supposedly by activeoxygen species. Furthermore, by sending it into the living body in a newdelivery form, namely by drip, it becomes possible to provide patientswith a serious illness or a brain disease, who are incapable of oralintake, with coenzymes Q. It is thus expected that solubilization ofcoenzymes Q will bring about a number of merits.

As is well known, coenzymes Q can occur in both the oxidized form andreduced form, and a number of investigations have so far been made aboutthe method of solubilizing oxidized coenzyme Q₁₀ (ubidecarenone orubiquinone).

As for the solubilization of oxidized coenzyme Q₁₀, various methods havebeen reported, for example coating with liposomes, suspension using asurfactant or an oil/fat, and the like (Japanese Kokai PublicationHei-05-186340, Japanese Kokai Publication Hei-07-69874, Japanese KohyoPublication 2000-510841). However, few examples have been put topractice use. One of the reasons is that while it is necessary foroxidized coenzyme Q₁₀ to be converted to the reduced form by the actionof a reductase or the like in order to perform its antioxidant activity,no reductase is present in blood and, therefore, no antioxidant activityagainst oxidative stresses in blood can be expected upon administrationby injection or the like.

On the other hand, reduced coenzyme Q₁₀ itself has antioxidant activity,hence it is a substance much expected to be of great utility in theprevention/treatment of such diseases as mentioned above. However, ithas not been put into practical use because of its drawback that it issusceptible to oxidation and thus unstable. Although a search reportdescribing the preparation of liposome-coated reduced coenzyme Q₁₀ forthe purpose of studying oxidoreductases and so forth is available (Kishiet al., 1999, BioFactors, 10, 131-138), the liposomes used were preparedextemporaneously in each experiment. Any method of stably solubilizingreduced coenzymes Q has not been known at all.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an aqueous solutionimproved in oxidation stability of reduced coenzymes Q.

The present inventors made investigations in an attempt to accomplishthe above object and, as a result, found out an aqueous solutioncomposition suitable for increasing the stability of reduced coenzymesQ, which has led to completion of the present invention.

Thus, the present invention provides an aqueous solution containing areduced coenzyme Q, which comprises an antioxidant and/or a chelatingagent in the same aqueous solution.

DETAILED DESCRIPTION OF THE INVENTION

The aqueous solution of the invention is an aqueous solution containinga reduced coenzyme Q represented by the following formula (1):

in the formula, n represents an integer of 1 to 12:

which comprises an antioxidant and/or a chelating agent in the sameaqueous solution.

The coenzyme Q includes the species represented by the following formula(1):

in the formula, n represents an integer of 1 to 12: and the speciesrepresented by the following formula (2):

in the formula, n represents an integer of 1 to 12. The formula (l)represents the reduced form of a coenzyme Q and the formula (2)represents the oxidized form of a coenzyme Q.

In the practice of the invention, the coenzyme Q to be used may be anyof those in which the number (n in the formulas) of repeating units inthe side chain is 1 to 12, as represented by the above formulas (1) and(2). However, the one having 10 repeating units in the side chain,namely coenzyme Q₁₀, can be used most adequately.

In the practice of the invention, an oxidized coenzyme Q may coexistwith the reduced coenzyme Q to be used. In that case, the content of thereduced coenzyme Q in the whole amount of coenzymes Q is preferably notless than 20% by weight, more preferably not less than 40% by weight,most preferably not less than 60% by weight.

The method of preparing the reduced coenzyme Q is not particularlyrestricted but, for example, the method comprising producing a coenzymeQ by any of the methods known in the art, for example by synthesis,fermentation, extraction from a natural source, or the like method andthen concentrating the reduced coenzyme Q fraction in the eluate bychromatography can be employed. In this case, it is also possible to adda conventional reducing agent, such as sodium borohydride or sodiumdithionite (hydrosulfite sodium), to the coenzyme Q if necessary and,after reduction of the oxidized coenzyme Q contained in the abovecoenzyme Q to a reduced coenzyme Q in the conventional manner, subjectthe reduction product to chromatography for concentration. It can alsobe obtained by the method comprising reacting a commercially availablehigh-purity coenzyme Q with such a reducing agent as mentioned above.

The method of preparing the aqueous solution of the invention is notparticularly restricted but, for example, the aqueous solution can beobtained (1) by coating a reduced coenzyme Q and an appropriateantioxidant and/or an appropriate chelating agent with an appropriateliposome base to give liposomes, (2) by adding an aqueous solution of anappropriate antioxidant and/or an appropriate chelating agent to reducedcoenzyme Q-containing liposomes, or (3) by solubilizing or emulsifying areduced coenzyme Q and an appropriate antioxidant and/or an appropriatechelating agent by means of an appropriate surfactant.

The aqueous solution of the invention is not particularly restricted butmay be any one containing a reduced coenzyme Q and an antioxidant and/ora chelating agent. When it is prepared using liposomes, as mentionedabove under (1) or (2), it occurs as an aqueous solution with theliposomes being dispersed in the aqueous solution. When it is preparedusing a surfactant, as mentioned above under (3), it occurs as anaqueous solution with a reduced coenzyme Q being solubilized oremulsified therein.

The antioxidant that can be used in the practice of the presentinvention is not particularly restricted but includes, for example,citric acid, citric acid derivatives, vitamin C, vitamin C derivatives,probucol, licopene, vitamin A, carotenoids, vitamin B, vitamin Bderivatives, flavonoids, polyphenols, glutathione, selenium, sodiumthiosulfate, vitamin E, vitamin E derivatives, superoxide dismutase(SOD), glutathione peroxidase, glutathione-S-transferase, glutathionereductase, catalase, ascorbate peroxidase and mixtures of these.

Among them, citric acid, citric acid derivatives, vitamin C, vitamin Cderivatives, glutathione and sodium thiosulfate are preferred, vitaminC, citric acid and the like are more preferred, and vitamin C is stillmore preferred.

The chelating agent is not particularly restricted but includes, forexample, ethylenediaminetetraacetic acid and salts thereof,ethylenediaminediacetic acid and salts thereof, hydroxyiminodiaceticacid and salts thereof, hydroxyethylethylenediaminetetraacetic acid andsalts thereof, diethylenetriaminepentaacetic acid and salts thereof,nitrilotriacetic acid and salts thereof, triethylenetetraminehexaaceticacid and salts thereof, dicarboxymethylglutamic acid tetrasodium salt,dihydroxymethylglycine, 1,3-propanediaminetetraacetic acid and saltsthereof, 1,3-diamino-2-hydroxypropanetetraacetic acid and salts thereof,sodium gluconate, hydroxyethylidenediphosphonic acid, alkylenephosphonicacids, phosphonobutanetricarboxylic acid and mixtures of these.

Among these, ethylenediaminetetraacetic acid and salts thereof,hydroxyethylethylenediaminetetraacetic acid and salts thereof,diethylenetriaminepentaacetic acid and salts thereof, sodium gluconateand hydroxyethylidenediphosphonic acid are preferred, andethylenediaminetetraacetic acid and salts thereof are more preferred.

In cases where liposomes are used in the aqueous solution of theinvention, the liposome includes, for example, phospholipids such asrefined soy lecithin and phosphatidylcholine, glicolipids such asdigalactosylglyceride, and the like. From the viewpoint of thestabilization of reduced coenzymes Q against oxidation, refined soylecithin, phosphatidylcholine and the like are preferred.

When a surfactant is used in the aqueous solution of the invention, thesurfactant is not particularly restricted but includes, for example,carboxylate salt type anionic surfactants and the like. Preferred fromthe viewpoint of the oxidation stability of reduced coenzymes Q arepolysorbate 80, polyoxyethylene hydrogenated castor oil and the like.

The reduced coenzyme Q concentration in the aqueous solution of theinvention is not particularly restricted but, from the viewpoint ofoxidation stability, solubility in aqueous solution, and/or the like,the proportion of the reduced coenzyme weight to the volume of theaqueous solution is preferably not lower than 0.001% (w/v) but nothigher than 5% (w/v), more preferably not lower than 0.05% (w/v) but nothigher than 1% (w/v) . The antioxidant concentration is not particularlyrestricted, either. From the efficacy viewpoint, however, it ispreferably not lower than 0.01% (w/v) but not higher than 50% (w/v),more preferably not lower than 0.05% (w/v) but not higher than 10%(w/v), relative to the whole amount of the aqueous solution. Thechelating agent concentration is not particularly restricted, either.From the efficacy viewpoint, however, it is preferably not lower than0.001% (w/v) but not higher than 10% (w/v), more preferably not lowerthan 0.005% (w/v) but not higher than 5% (w/v), relative to the wholeamount of the aqueous solution. The antioxidant concentration andchelating agent concentration mentioned above are applicable not only inthe case where these are respectively used singly but also in the caseof combined use thereof.

The pH of the aqueous solution of the invention is not particularlyrestricted but may vary according to the intended use thereof. From theviewpoint of the stability of coenzymes Q, however, the pH is preferablynot lower than 1.0 but not higher than 8.0, more preferably not lowerthan 2.0 but not higher than 7.6.

To the aqueous solution to be prepared in the above manner, there may beadded one or more other pharmaceutically acceptable preparationmaterials each at an appropriate addition level in the conventionalmanner. The preparation materials are not limited but there may bementioned, for example, emulsifiers, isotonizing agents, buffers,solubilizing agents, correctives for smell, preservatives, stabilizersand the like. Furthermore, a further active ingredient, for example adrug and/or a nutritional supplement, may be added with the intendeduse.

The method of storing the aqueous solution composition according to theinvention is not particularly restricted but includes cold storage (e.g.at −80° C. to 4° C.), anaerobic storage in a tightly closed container,and storage under protection from light, and the like.

The aqueous solution of the invention which can be prepared in the abovemanner can preserve reduced coenzymes Q more stably from oxidation.

The reduced coenzyme Q-containing aqueous solution according to theinvention can be used in a wide range, including medical, cosmetic,food, horticultural, and dairy uses, and the like. As specificpreparation forms, there may be mentioned injectable solutions, infusionsolutions, liquid preparations, ophthalmic solutions, solutions for oraladministration, lotions, hair tonics, milky lotions, sprays, aerosols,health drinks, liquid fertilizers, preserving solutions and so on. Inthe field of medicine, it can further be used as a preserving solutionon the occasion of organ transplantation. Furthermore, the use as animalfeeds and so on may also be mentioned. In addition, it can be used, asan antioxidant solution, in storing meat, fish and other perishablefoods.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail. These examples are, however, by no means limitative of the scopeof the invention.

EXAMPLE 1

Oxidation Stability of Liposome-Coated Reduced Coenzyme Q₁₀ as Given byan Antioxidant

For evaluating the oxidation stability of reduced coenzyme Q₁₀ inliposomes, yolk lecithin (lecithin, product of Wako Pure ChemicalIndustries), which has poor oxidation stability, was selected as theliposome base, and the evaluation of substances capable of increasingthe oxidation stability of reduced coenzyme Q₁₀ (content 0.005% w/v) wasperformed under the storage condition of 40° C.

Reduced coenzyme Q₁₀-containing liposomes were prepared by the followingmethod. Thus, a powder of reduced coenzyme Q₁₀ was dissolved in ethanolto give a 0.1 mg/ml solution. Similarly, a lecithin solution (3.2 mg/ml)in ethanol was prepared. Both solutions (1 ml each) were mixed together.To the resulting mixture was added 2 ml of chloroform and, then, thesolvent was removed under reduced pressure. After complete eliminationof the solvents, 2 ml of a 50 mM HEPES buffer solution (pH 7.4) wasadded, and the lipid film was dispersed using a mixer to give amilk-white suspension. This suspension was subjected to sonication in anitrogen atmosphere at 4° C. for 30 minutes for the formation ofliposomes, followed by 20 minutes of centrifugation at 3,000 rpm toremove giant molecules as a sediment. The fraction remaining unsettledafter centrifugation was used as reduced coenzyme Q₁₀-containingliposome solution.

Liposomes containing an antioxidant or a chelating agent (0.05% w/v)were prepared as follows. In the case of vitamin E, a 10 mg/ml ethanolsolution was prepared and added to a mixed solution containing reducedcoenzyme Q₁₀ and lecithin prior to the addition of chloroform in theabove-mentioned process for preparing reduced coenzyme Q₁₀-containingliposomes and, thereafter, liposomes were prepared in the same manner asmentioned above. In the case of other water-soluble antioxidants orchelating agents, each antioxidant or chelating agent was dissolved inthe above-mentioned HEPES buffer solution to a concentration of 0.05%(w/v) and, thereafter, liposomes were prepared in the same manner.

In this manner, liposomes containing various antioxidant (vitamin Eacetate, sodium thiosulfate, citric acid, vitamin C, vitamin E(α-tocopherol)) were prepared and evaluated for oxidation stabilityduring storage at 40° C. in the ambient atmosphere. The results wereexpressed in terms of residual reduced coenzyme Q₁₀ percentage.

For determining the residual reduced coenzyme Q₁₀ percentage, 1 ml ofhexane was first added to 0.05 ml of each liposome solution and, after30 seconds of stirring, the mixture was separated into a hexane layerand an aqueous layer by centrifugation (3,000 rpm, 1 minute). Then, thehexane layer was recovered and evaporated to dryness in a nitrogenatmosphere, the residue was dissolved in 0.2 ml of ethanol, and thesolution was subjected to assaying by HPLC. The HPLC was carried outunder per se known analytical conditions using an electrochemicaldetector, and oxidized coenzyme Q₁₀ and reduced coenzyme Q₁₀ wererespectively quantitated. The residual reduced coenzyme Q₁₀ percentagewas calculated as a percentage relative to the amount of reducedcoenzyme Q₁₀ on the occasion of preparing liposomes.

As a result, the antioxidant-free liposomes underwent oxidation in sucha manner that the residual percentage lowered to 10% or below during 1to 2 days of storage, whereas the residual percentage was 63% after 2days of storage in the case of vitamin E acetate-containing liposomes,76% after 3 days of storage in the case of sodium thiosulfate-containingliposomes, 54% after 8 days of storage in the case of citricacid-containing liposomes, or 68% after 15 days of storage in the caseof vitamin C-containing liposomes. However, the addition of vitamin E(α-tocopherol) failed to produce any remarkable effect.

It was thus found that some antioxidants are strongly effective andothers are mildly effective in stabilizing reduced coenzyme Q₁₀ inliposomes. More specifically, vitamin C was the most effective, followedby citric acid, sodium thiosulfate, vitamin E acetate, and vitamin E inthat order.

EXAMPLE 2

Oxidation Stability of Liposome-Coated Reduced Coenzyme Q₁₀ as Given bya Chelating Agent

The effect of a chelating agent on the residual reduced coenzyme Q₁₀percentage was evaluated in the same manner as in Example 1. Liposomescontaining 0.05% (w/v) of ethylenediaminetetraacetic acid were preparedin the same manner as in Example 1 and stored in the air at 40° C. forevaluating the stabilizing effect of the chelating agent.

The reduced coenzyme Q₁₀ in chelating agent-free liposomes was oxidizedto a residual percentage of 10% or below during 1 to 2 days of storage,whereas the ethylenediaminetetraacetic acid-containing liposomes showeda residual percentage of 76% after 43 days of storage.

It is known in the art that the chelating agentethylenediaminetetraacetic acid is more or less effective in protectingsubstances susceptible to oxidation. However, the finding obtained inthis example that it is comparable or superior in such protective effectto antioxidants was quite unexpected.

EXAMPLE 3

Effect of an Antioxidant on a Surfactant Solution—(1)

To a 0.1% aqueous polysorbate 80 solution containing 0.005% (w/v) ofreduced coenzyme Q₁₀ was added vitamin C, as an antioxidant, to aconcentration of 0.05% (w/v), the solution was stored at 40° C., and theeffect of the antioxidant was evaluated.

The solution without addition of the antioxidant gave a residualpercentage of 27% after 6 days of storage. On the contrary, 88% of thereduced form was retained in the vitamin C-containing solution evenafter 6 days of storage.

EXAMPLE 4

Effect of an Antioxidant on a Surfactant Solution—(2)

To a solution containing 0.1% of polyoxyethylene hydrogenated castor oil(HCO-60; product of Nikko Chemicals) and 0.005% (w/v) of reducedcoenzyme Q₁₀ was added vitamin C or citric acid, as an antioxidant, to aconcentration of 0.05% (w/v), the solution was stored at 40° C., and theeffect of the antioxidant was evaluated.

The solution without addition of the antioxidant gave a residualpercentage of 17% after 7 days of storage. On the contrary, 73% of thereduced form was retained in the vitamin C-containing solution evenafter 14 days of storage, and 61% of the reduced form was maintained inthe citric acid-containing solution even after 71 days of storage.

EXAMPLE 5

Effect of a Chelating Agent on a Surfactant Solution—(1)

The effect of a chelating agent was evaluated in the same manner as inExample 3 except that ethylenediaminetetraacetic acid was used as thechelating agent in lieu of the antioxidant.

The solution without addition of the chelating agent gave a residualpercentage of 27% after 6 days of storage, whereas 80% of the reducedform was retained in the ethylenediaminetetraacetic acid-containingsolution even after 78 days of storage, indicating the same potentprotective effect as in the case of liposomes.

EXAMPLE 6

Effect of a Chelating Agent on a Surfactant Solution—(2)

The effect of a chelating agent was evaluated in the same manner as inExample 4 except that ethylenediaminetetraacetic acid was used as thechelating agent in lieu of the antioxidant.

The solution without addition of the chelating agent gave a residualpercentage of 17% after 7 days of storage, whereas 74% of the reducedform was retained in the ethylenediaminetetraacetic acid-containingsolution even after 167 days of storage. The result obtained thatreduced coenzyme Q₁₀ can be maintained at high residual percentagesunder such high temperature conditions as 40° C. over a period as longas nearly half a year was quite unexpected.

INDUSTRIAL APPLICABILITY

In accordance with the invention, liquid preparations containing reducedcoenzymes Q, which are highly useful as antioxidant substances ornutritional supplement components, can be provided more stably.

1. An aqueous solution containing a reduced coenzyme Q represented bythe following formula (1):

in the formula, n represents an integer of 1 to 12: which comprises anantioxidant and/or a chelating agent in the same aqueous solution.
 2. Anaqueous solution according to claim 1, wherein the reduced coenzyme Q isreduced coenzyme Q₁₀.
 3. An aqueous solution according to claim 1,wherein the chelating agent is ethylenediaminetetracetic acid or a saltthereof, hydroxyethylethylenediaminetetraacetic acid or a salt thereof,diethylenetriaminepentaacetic acid or a salt thereof, sodium gluconateor hydroxyethylidenediphosphonic acid.
 4. An aqueous solution accordingto claim 1, wherein the antioxidant is citric acid, a citric acidderivative, vitamin C, a vitamin C derivative, glutathione or sodiumthiosulfate.
 5. An aqueous solution according to claim 2, wherein thechelating agent is ethylenediaminetetracetic acid or a salt thereof,hydroxyethylethylenediaminetetraacetic acid or a salt thereof,diethylenetriaminepentaacetic acid or a salt thereof, sodium gluconateor hydroxyethylidenediphosphonic acid.
 6. An aqueous solution accordingto claim 2, wherein the antioxidant is citric acid, a citric acidderivative, vitamin C, a vitamin C derivative, glutathione or sodiumthiosulfate.